Biological Upgrading of SugarsWBS 2.3.2.105

Principal Investigator: Gregg T. BeckhamOrganization: National Renewable Energy Laboratory

This presentation does not contain any proprietary, confidential, or otherwise restricted information.

2017 DOE Bioenergy Technologies Office (BETO) Project Peer ReviewDate: March 7, 2017Technology Area Review: Biochemical Conversion

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Goal StatementGoal: Develop industrially-relevant strains to meet titer, rate, and yield targets for fuel precursors for the 2022 BC Platform cost target goals of $3/GGE• Focus on aerobic fatty acids and anaerobic, secreted C2-C6 carboxylic acids

Relevance: Success could enable eventual commercial adoption of these routes and associated strains

HC fuels will be a major benefit to the U.S. biorefinery infrastructure.• Conduct TEA/LCA to identify cost drivers and data gaps and to refine process options.• Collaborate with national labs, academics, and industry for strain development and process demonstration.• Outcome: Robust strains to meet titer, rate, and yield targets for producing HC fuels precursors.

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Quad Chart

• Start date: 10/14

• End date: 09/17

• Percent complete: 83%

• Ct-H: Efficient Cat. Upgrading of Sugars– Strain development for titer, rate, yield targets

• Ct-J: Process Integration– Employing hydrolysate in bioreactors and working

with upstream and downstream partners• Ct-G: Efficient Intermediate Cleanup

Timeline

Budget

Barriers

• NREL BETO projects: Biochemical Platform Analysis; Bench-Scale Integration; Separations Development and Application; Catalytic Upgrading of Sugars; Pretreatment and Process Hydrolysis; Pilot-Scale Integration; Biochemical Process Modeling and Simulation; Strategic Analysis Platform

• BETO-funded national lab projects: Agile BioFoundry, Separations Consortium, Catalytic Upgrading of Biochemical Intermediates

• Industrial collaborators: Fondevila NeolBio, Earth Energy Renewables

• Academic collaborators: Colorado State University; University of Pretoria; MIT; Penn State University; Temasek Life Sciences Laboratory; Università degli Studi di Milano-Bicocca

Partners and Collaborators

FY15 FY16

Total Planned Funding (FY17)

DOE-funded $1,800K $1,800K $1,800K

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Process integration, Scale-up, Verification

Process Development

Synthesis and Upgrading Technol.

Enabling/Fundamental Technologies

Feedstock-Process Interface Pretreatment

and Process Hydrolysis

Separations Development

and Application

Enzyme Engineering and

Optimization

Biological Upgrading of

Sugars Bench-Scale Process

Integration

Pilot-Scale Process

Integration

Biochemical Platform Analysis

Targeted Microbial

Development

Biochem. Process Modeling and

Simulation

Lignin Utilization

Catalytic Upgrading of Biochem.

IntermediatesBiological Lignin Depolymerization

Analytical Development and Support

Interactions with other BC Conversion Projects

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Project Overview

Context: “Beyond ethanol” to produce a portfolio of biofuels• Produce direct replacements or

blendstocks for diesel and jet fuel• Shifted to intermediates that can be

upgraded to diesel and jet fuel

Project Objectives:- Develop industrially relevant strains for fatty acids and short-chain carboxylic acids to

meet 2022 cost targets. - Focus efforts toward titer, rate, and yield targets set by TEA/LCA modeling.- Rapidly test strains with Bench-Scale Integration to identify and solve problems in scaling.

Davis et al., NREL Design Report, 2014

History: HC fuel R&D primarily began at NREL in the Nat’l Adv. Biofuels Consortium• TEA suggests coproducts essential to cost-effective HC production • NREL developing plans after 2012 to meet 2022 targets for $3/GGE HC fuels • BUS project began in FY15

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Technical Approach

Approach:• Target: 1 g/L/h rate, 60% lipid content, and a

0.27 g/g yield on hydrolysates• Screen naturally oleaginous strains.• Engineer strains for secretion or easier lysis.

Primary challenges and success factors:• High yield and productivity of lipids• Availability of genetic tools in strains for

metabolic engineering• High cost for cell lysis• High cost for aerobic processing

Aim 1: Develop a robust oleaginous strain

Approach: • Target: >95% sugar utilization, ≥2 g/L/h rate,

≥80% of theoretical yield on hydrolysates• Screen yeast and bacteria strains on

hydrolysates.• Engineer a strain for higher yields.

Primary challenges and success factors:• Overcoming hydrolysate toxicity.• Increasing carbon flux to target acids over

side products (in bacteria).• Incomplete sugar utilization (in yeast)

Aim 2: Develop robust C2 and C4 carboxylate strains

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Management Approach

Approach:

• Define optimal strain attributes based on performance in bioreactors with hydrolysate

• Down-select optimal strains (e.g., oleaginous, low pH, or effective carboxylate producer)

• Develop genetic tools (if none exist)

• Define targets in collaboration with TEA, LCA, and downstream processing (separations and catalysis)

• Improve other strain attributes (e.g., knockout by-products, increase sugar utilization capacity, target a different product than native metabolism)

• Evaluate engineered strains in bioreactors on hydrolysate

Experienced task leads in fermentation, microbiology, and metabolic engineering

Milestones prioritized to down-select single strains for 2022 deployment

Screen for optimal strains

Conduct strain engineering

Adapt/probe tolerance

mechanisms

Evaluate engineered

strains

Integrate with separations and catalysis

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Systematic screen of 32 oleaginous yeast strains

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• Titer, rate, yield• Lipid classing• Oil extraction• Detoxification mechanisms• Oil upgrading

Bioreactor performance on hydrolysate

FAME: %, Q and titer32 strains

Rt 4444Top performersRhodosporidium toruloidesCryptococcus curvatusTrichosporon guehoae

Outcome: Down-selected R. toruloides 4444 as a robust oleaginous strain in hydrolysate that is also genetically tractable; fed-batch optimization ongoing

Shake flasks

Hydrolysate100 g/L sugars

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Engineering R. toruloides

Bright GFP expression

Generatedexpression plasmids

Transformations viaconjugation and electroporation

Polycistronic expression constructs

In partnership with:

Fatty Alcohols

DSM4444 4444 9-6

*NSL134: 8 g/L fatty alcohols (Fillet et al. 2015)

*0200400600800

100012001400160018002000

N1234 4444 9-6

Fatty

Alc

ohol

s (A

U)

1-Eicosanol

11-Eicosenol

1-Octadecaol

Octadecenol

1-Hexadecanol

Hexadecenol

Fatty alcohols present in Rhodosporidium strains

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Enhancing cell lysis

Outcome: Determined 77 non-obvious targets for enhancing cell lysis in oleaginous yeast

Hyper-sensitivemutant

Slightlysensitivemutant

Intrinsicallyweakmutant

Control Plus T. harzianumlytic enzyme

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Shifting priorities to nearer term pathways

Oleaginous pathways at $3/GGE:- 1 g/L/h, 70% lipids, 0.27 g lipids/g total

sugars, 100% glucose, 85% C5 utilization- $5/GGE contribution from lignin - 95% lipid recovery via auto-lysis- Scale of aerobic << anaerobic - Produces straight-chain HCs

Anaerobic acid pathway at $3/GGE:- 100% glucose utilization, 85% C5

utilization, 1.5-day fermentation- $2.50/GGE contribution from lignin- Either extractive or low-pH fermentation- Can produce branched diesel and jet

fuel-range HCs

C2-C4 acids are ideal fuel precursors- Same theoretical yield as EtOH- Can produce branched fuels

R. Davis et al. 2016 NREL Milestone

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Strains selection based on• C5/C6 sugar utilization• Anaerobic production of C2-C4 acids• Tolerant of inhibitors in hydrolysate• Genetically tractable• Screened multiple anaerobic bacteria

Clostridium butyricum: genetically tractable, grows in biomass hydrolysate, produces C2 and C4 acids, utilizes all sugars efficientlyC. tyrobutyricum: genetically tractable, grows in biomass hydrolysate, produces AA and BA, poor xylose and arabinose utilization

C. tyrobutyricum C. butyricum

Bacterial screening for carboxylate production

C4 C2Titer (g/L) 20.7 10.5Yield (g/g) 0.3 0.15Max. productivity (g/L/h) 1.6 0.8

C. butyricum on hydrolysate

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Arabinose

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Clostridium butyricum metabolic engineering

Target increased flux to acetate and butyrate with enhanced carbon efficiency• Overexpression of C2 and C4

acid biosynthetic machinery• Knockout of competitive carbon

pathways (lactate, formate, EtOH)• Bypass of pyruvate

decarboxylation via homologous overexpression of native phosphoketolase pathway

Adapted from Yu et al. 2011. Metabolic Engineering 13(4), 373–382.

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Baseline extraction• Used a C4 and C6 acid-

producing strain “off-the-shelf” to develop baseline extraction

• Working with the Sep. Consortium to develop extraction methods to recover acids

MembraneContactorsPeristaltic

Pump

PressureGauge

Valve

Organic phase NaOH

Feed

Aqueous phase

Extractive fermentation • No pH control• Process intensification• Decreased product

toxicity • Increased C4 extraction

efficiency• Continuous feeding

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Acetic AcidButyric AcidHexanoic Acid

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BA 32 g/L

HA 20 g/L

AA 2 g/L

Demonstrating extractive fermentation

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Zygosaccharomyces bailii:• Acid tolerant (pH 2, high concentration of

organic acids)• Osmotolerant (can grow up to 90% sugars)Pichia kudriavzevii:• Engineered for malonic, lactic, and succinic

acids, along with xylonate• Acid tolerant (pH 2–3)Candida glabrata:• Effective pyruvic acid producer• CRISPR-Cas9 tools developed• Acid tolerant (can grow at pH 2)

Yeast could enable low-pH carboxylic acid production

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P. kudriavzeviiZ. bailii

Anaerobic hydrolysate performance (3 strains):• High metabolic EtOH yields • Able to ferment hydrolysate w/o extra nutrients• Engineering planned for C5 sugar utilization

Zb Pk CgTiter (g/L) 41.9 45.0 38.5Yield (g/g) 0.42 0.49 0.45Max. productivity (g/L/h) 0.36 0.27 0.31

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C. glabrata

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Adapted from Yu, et al., 2011 Metabolic Engineering 13(4), 373–382.

Low-pH yeast metabolic engineering targets

Leverage existing genetic tools to:• Heterologously express C2 and C4 acid

biosynthetic machinery.

• Bypass pyruvate decarboxylation to increase carbon efficiency by overexpression of the phosphoketolase (pkt) pathway.

• Integrate xylose assimilation pathway.

• Heterologously express alcohol acetyl transferases (AATs) to generate ethyl acetate and ethyl butyrate, in non-pkt-overexpressed strains.

Adapted from Yu et al. 2011. Metabolic Engineering 13(4), 373–382.

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RelevanceKey MYPP areas for the Biological Upgrading of Sugars project:

Efficient Upgrading of Sugars- Developing efficient biocatalysts

to produce advanced fuels and fuel precursors

- Improving titer, rate, yield key to economic viability

Biochemical Conversion Process Integration- Coupling process considerations

with organism development- Working with BSI task to iterate

on fermentation needs and organism modifications/evolution

Efficient Clean-up and Conditioning- Elucidating inhibitor effects on

biocatalysts and downstream processing

Key Stakeholders and Impacts- Research focused on carbohydrate utilization in

HC fuel production from biomass

- Portfolio of potential oleo- and carboxylate-based fuels and chemicals will diversify and accelerate the biomass value chain

- Methods to upgrade sugars to organics acids can be leveraged well beyond fuel precursors

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Future Work

Oleaginous yeast:- Transition away from this work because of

high process cost for producing diesel- Hand off strains and tools to longer term

strain development in the Targeted Microbial Development project

Anaerobic bacteria platform:- Down-selected to C. butyricum- Engineer improved flux to C2 and C4

products- Integrate with separations and catalysis

Anaerobic low-pH yeast platform:- Performing down-selection now- Improve C5 sugar utilization- Target C2 and C4 acids requiring no online

extraction (low-pH fermentation)

Metabolomics, transcriptomics, and proteomics to baseline performance

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Future Work

Strain engineering will require a $1.25/GGE improvement from the current SOTCarboxylates are efficient intermediates that can enable production of jet fuel and dieselClostridium butyricum:• Intensify fermentation and separations for higher titer, rate, yield toward targets (1.5-day

fermentation, >95% sugar utilization, no pH control extraction)o 2-day fermentation, ~90% sugar utilization, extraction with other microbes successful so far

• Maximize carbon flux to C2 and C4 acids or easily separable products (e.g., esters)Low-pH yeast:• Expand sugar utilization to include pentose sugars• Divert more carbon to fuels through more efficient strains

Aim: Single strain and associated process by end of FY19 for scaling

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Summary1) Approach:

• Develop oleaginous yeast for lipid production for renewable diesel blends • Develop carboxylate fuel precursor strains for C2/C4 acids to produce branched jet/diesel blendstocks

2) Technical accomplishments • Screened a large collection of oleaginous yeast, down-selected R. toruloides• Identified multiple mechanisms of inhibitor tolerance in aerobic oleaginous yeast• Identified 77 gene targets for enhancing cell lysis in oleaginous yeast strains• Developed fatty alcohol strains of R. toruloides• Demonstrated near-theoretical yields of C2/C4 acids in C. butyricum and evaluated low pH yeast

strains for anaerobic growth, both on biomass hydrolysates• Developed bench-scale extractive fermentation capabilities• Metabolic engineering underway for bacteria (C. butyricum) and will start soon in low pH yeast

3) Relevance• Directly impacts the 2022 HC fuel cost target demonstrations through strain development• Addresses Whole Barrel of Oil Initiative and bolsters the biomass value chain

4) Critical success factors and challenges• Economic and sustainable production of fuel precursors, rapid tool development and integration with

downstream processes5) Future work:

• Continue towards down-selection of a carboxylate strain for 2022 demonstration6) Technology transfer:

• Working with industry to build commercialization path for oleaginous yeast pathways, developing a broad IP portfolio on engineered carboxylate strains

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Acknowledgments• Jeffrey Linger• Andrew Lowell• Ali Mohagheghi• Bill Michener• Robert Nelson• Darren Peterson• Kelsey Ramirez• Michelle Reed• Michael Resch• Davinia Salvachua• Violeta Sanchez i

Nogue• Maria Sanchez• Christine Singer• Emily Singer• Priyanka Singh• Thieny Trinh• Melvin Tucker• Neil Wagner• Xiaoqing Wang• Rou Yi Yeap• Xiunan Yi

External collaborators• Ken Reardon and Xingfeng Huang, Colorado

State University• Willie Nicol, University of Pretoria• Catie Bartlett, Laura Crowell, Troy Langford,

Daniel Salem, Akshar Wunnava, School of Chemical Engineering Practice, MIT

• Samuel Purvine and Erika Zink, EMSL• Jose Luis Adrio, Fondevila NeolBio• Paola Branduardi, Università degli Studi di

Milano-Bicocca• Ji Liang Hui, Temasek Life Sciences Laboratory,

Singapore

• BETO: Jay Fitzgerald and Brandon Hoffman

• Mary Biddy• Brenna Black• Michael Bradfield• Adam Bratis• Xiaowen Chen• Yat-Chen Chou• Nicholas

Cleveland• Ryan Davis• Nancy Dowe• Rick Elander• Mary Ann Franden• Emily Fulk• Michael Guarnieri• Calvin Henard• Eric Knoshaug• Jacob Kruger

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PublicationsPublications in press:1. J.D. McMillan and G.T. Beckham, “Thinking big: Towards ideal strains and processes for large-scale aerobic biofuels production”, in press at Microbial Biotech.,

invited Crystal Ball Feature.2. D. Salvachúa, H. Smith, P.C. St. John, A. Mohagheghi, D.J. Peterson, B.A. Black, N. Dowe, G.T. Beckham, “Succinic acid production from lignocellulosic

hydrolysate by Basfia succiniciproducens”, Biores. Tech. (2016) 214, pp. 558-5663. M.J. Biddy, R. Davis, D. Humbird, L. Tao, N. Dowe, M.T. Guarnieri, J.G. Linger, E.M. Karp, D. Salvachúa, D.R. Vardon, G.T. Beckham, “The techno-economic basis

for coproduct manufacturing to enable hydrocarbon fuel production from lignocellulosic biomass”, ACS Sust. Chem. Eng. (2016) 4(6), pp. 3196-3211.4. D. Salvachúa, A. Mohagheghi, H. Smith, M.F.A. Bradfield, B. Black, W. Nicol, M.J. Biddy, N. Dowe, G.T. Beckham, “Succinic acid production on xylose-enriched

biorefinery streams by Actinobacillus succinogenes in batch fermentation”, Biotech. Biofuels. (2016) 9, 28.5. M.F.A. Bradfield, A. Mohagheghi, D. Salvachua, H. Smith, B.A. Black, N. Dowe, G.T. Beckham, W. Nicol, “Continuous succinic acid production by Actinobacillus

succinogenes on xylose-enriched hydrolysate”, Biotech. Biofuels. (2015) 8, 181.

Publications in review1. R. Nelson, D. Peterson, E.M. Karp, G.T. Beckham, D. Salvachúa, “Mixed carboxylic acid production by Megasphaera elsdenii from glucose and lignocellulosic

hydrolysate”, in review as an invited article at Fermentation.2. M.T. Guarnieri, Y.C. Chou, D. Salvachua, A. Mohagheghi, P. St. John, Y.J. Bomble, G.T. Beckham, “Metabolic engineering of Actinobacillus succinogenes for

enhanced succinic acid production”, in review at AEM.

Publications in preparation:1. J.G. Linger et al., “Enhanced cell lysis for microbial lipid production”2. V. Sànchez i Nogué, B.A. Black, J.S. Kruger et al., “Microbial lipid production, separations, and catalytic upgrading to renewable diesel”3. E.M. Karp et al., “Separation of high purity succinic and propionic acids from hydrolysate fermentation broth”4. D.R. Vardon, E.M. Karp, D. Salvachúa, N.A. Rorrer, et al. “Integrated production of succinic acid, 1,4-butanediol, and polybutylene succinate from lignocellulosic

biomass”

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Previous Reviewer CommentsProject overview• The project performers have communicated the projects history, the context in which the project fits into the portfolio, and its

high level objectives.• Biological upgrading of sugars; C5 and C6 derived products. two separate streams "going beyond ethanol" to produce a broad

portfolio of biofuels and biobased chemicalsTEA suggests co-products are required for hydrocarbon production

• Good project overview, although it's starting to look like every project is utilizing the alkaline-pretreated material in favor of the more commonly seen (commercially) acid-pretreated material. While the project clearly fits into some aspects of the BETO portfolio, it would be good to see the project history and background show a drive to improve economics/viability of technologies closer to commercialization.

• Good overview, looking for strains for C5 --> chemical, C6 --> fuel.• HC fuels and coproducts strains. Great context and overview and rational for project• Last october start - goal 2017 produce $5 gge. Alkaline - acid – hydrolysis.• Lipids and succinic acid.• Coproduct focus is a good focus.• Collaboration with other national labs and academics.• Strains for fatty acid and co-products.

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Previous Reviewer CommentsProject approach• The project performers have implemented technically sound research, development, and deployment approaches, and have

demonstrated the results needed to meet their targets.• The project performers have identified a project management plan that includes well-defined milestones and adequate

methods for addressing potential risks.• The project performers have clearly describe critical success factors which will define technical and commercial viability, and

that they have explained and understand the challenges they must overcome to achieve success.• Oleaginous yeast - fuel (lipids) and succinic acid from C5 stream.• Bench scale integration.• Combination of an oleaginous yeast to produce a hydrocarbon (lipid) and succinic acid as a value-add coproduct from the C5

stream.• Good approach. Clearly demonstrated benefit of driving C5 coproduct generation. Very clear, quantified goals.• Looking for natural strains, evolved strains and engineering of strains, good approach.• Oleaginous yeast strain- clear targets and overview of approach• SA strain - clear targets and overview of approach• Task and task leaders identified.• Nice flow chart to visualize relation of tasks and approach.• targets are quantifiable, challenges outlined, develop 2 strains for the two products/fuel.• Schedule driven to hit 2017.• Risk mitigation considered in strain selection and development C5 and C6 effective strains.

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Previous Reviewer CommentsTechnical Progress and Accomplishments• The project performers have made progress in reaching their objectives based on their project management plan.• The project performers have described their most important accomplishments in achieving milestones, reaching technical

targets, and overcoming technical barriers.• The project performers have clearly described the progress since the period of the last review.• Many strains being tested; Lipomyces is an example, high lipid content, also consumes C5 sugars

Cereulenin mutagenesistargets in oleaginous yeast pathways also working in Saccharomyces as a model - rapid screening tools for increasing fatty acid biosynthesis

• Steel in the ground to make starch based succinic, a number of strains evaluated for C5 sugars, two anaerobes and CO2 fixers, achieving high titers already

• Very good progress is being made to date.• Although this is a new project, just getting off the ground, significant progress has been made. Clear explanation of why work

done to date has been done, with down-selection justified clearly, as well. Excellent use of the Death Star in presentation. Clearly in the market for collaborators/etc to help with moving forward, this matches with other highly-integrated work and enhances probability of success.

• Good results to date on biomass sugars.• Good performance of oleaginous yeast on C6 sugar hydrolysates

Discovery and engineering of oleaginous yeasts - good approaches and delivering results• SA coproduct - logical selection of strains• FA results

.20 g/g sugar to product yield from hydrolyzate sugars looks promising• C to FA synthesis pathway focus• Using S cerevisiae is really understood and seems like a good place to focus the research.• Succinic acid C5 development using lignocellulosic C5's as feed.• Literature search initial selection of strains - 2 strains selected

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Previous Reviewer CommentsProject relevance• The project performers have describes how the project contributes to meeting Program/Technology Area goals and objectives

and the Bioenergy Technologies Office, as cited in the MYPP.• The project performers have considered applications of their expected outputs.• The project performers have presented the relevancy of this project and how successful completion of the project will advance

the state of technology and impact the viability of commercial bioenergy applications.• Just getting started; essential to hit $5/gge target• Very closely linked to other efforts• Coordinated with cleanup and separation• Co-products required for meeting economics• Very relevant program since it is well accepted that value add co-products are required to meet fuel economics for hydrocarbon

production.• Project clearly works toward 2017 hydrocarbon fuel cost targets. Catalyst efficiency, process integration, and separations are

target areas, supporting portfolio objectives. The project has clearly considered applications of their expected outputs. Success will have a significant impact on the state of the technology.

• Given the program targets for 2017/2022 this is very important to understand organisms that can work in the pretreated hydrolyzates and enzyme sugars to make succinic and lipids.

• High impact and alignment with BETO goals. Good linkage and coordination with other activities.• Catalyst efficiency, biochemical integration, and separation all part of program goals.

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Previous Reviewer CommentsFuture work• The project performers have outlined adequate plans for future work, including key milestones and go/no go decision points

through September 30, 2016.• The project performers have communicated key planned milestones and addressed how they plan to deal with upcoming

decision points and any remaining issues.• Down select to smaller number of strains in the future.

will need to develop routes to multiple chemicals in the future since the market sizes are vastly different• Future work is clearly described, relevant, and includes SMART goals. Clear explanation of how future work will support move

away from use of lignin in CHP. Impacts "whole barrel" initiative.• Good milestones and plans for succinic and lipid fermenting organisms.• Clear plan for selecting and improving strains. Quantitative targets for performance.• Down select strains to focus on.• Evaluate a different kind of pretreatment chemicals/mechanical?

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Previous Reviewer CommentsOverall Impressions (Not Scored)• This is a very relevant program due to the poor theoretical yields of hydrocarbons from sugars (resulting in poor

economics). The co-production of a value added chemical will aid in the overall economics, however there are potential market size issues. The market size of succinic acid is far smaller than hydrocarbons, therefore one may saturate the chemicals marketquickly which may limit the size of the hydrocarbon end of the process. Thus this may require multiple co-products be made instead of one. Secondly the capital and operating costs of a "split stream" biomass conversion process may be more than combined approach. This should be monitored.

• This is a good project, but almost seems to be two parallel projects working toward a common goal. Utilization of C5 for succinic acid and C6 for lipid production by a different organism raises questions as to commercial viability, as it's moving down a path long-travelled by unsuccessful projects. Separation of the streams increase capital costs, footprint, and complexity, but the inclusion of value-added products in the process (such as those produced from this project) may offset those issues. Focus should be on making chemical precursors in lieu of products that have the potential to flood the market immediately upon commercial scale application, since the scale economics may well be driven by C6 utilization.

• With the program process set as it is and succinic and lipids selected as the products of choice, this is an excellent task.• Very well conceived and planned project for converting biomass sugars into hydrocarbons. Selection of strains and target

molecules is clear and methodical. Some of the strains and approaches are innovative and promise to deliver some new options for hydrocarbon production.

• Interesting research, the fuel/chemical combination key.

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C. curvatus 20509 T. guehoae 60-59 R. toruloides 4444

• Aromatics as C source

• Oil upgrading (separations and catalysis)

Evaluation of oleaginous yeasts

p-Coumaric acid Ferulic acid

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